Genipin-cross-linked Scaffolds Influence The Differentiation Of Adipose-derived Stem Cells And Nucleus Pulposus Regeneration

Part 1:Genipin-cross-linked type ? collagen scaffold promotes the differentiation of adipose-derived stem cells into nucleus pulposus-like cellsObjectives:Adipose-derived stem cells(ADSCs)-based tissue engineering was a promising method to treat intervertebral disc degeneration.Type II collagen is a native component in the nucleus pulposus(NP),and has the ability to promote ADSCs to differentiate into NP-like cells.In this article,we aimed to establish a genipin-cross-linked three-dimensional(3D)type ? collagen scaffold,and determine the biological effects of the scaffold on ADSCs differentiating into a NP-like phenotype.Methods:Different concentrations of genipin were used to cross-link the 3D type ? collagen scaffold.Microstructure,surface topography,mechanical strength,porosity,swelling property,and biological stability of the scaffolds were detected to evaluate the scaffold properties.Cell proliferation,gene and protein expression were measured to access the biological effects of the scaffolds on ADSCs,and the related molecular mechanism was investigated.Results:As the concentration of genipin increased from 0.1 to 1%,the surface of the type ? collagen scaffolds became rougher and an increasing amount of granular uplift could be observed.After cross-linking with glutaraldehyde,the scaffold surface became rough and demonstrated flake uplift.The Young's modulus of the 3D type ? collagen scaffold without cross-linking was 1.196 ± 0.262 MPa.When cross-linking was performed with0.1% genipin,the Young's modulus increased to 2.326 ± 1.123 MPa.When the concentration of genipin increased to 0.5% and 1%,the Young's modulus increased rapidly to 12.517 ± 2.652 and 19.531 ± 4.264 MPa,respectively.Before cross-linking,the porosity of the 3D type ? collagen scaffold was 95.673 ± 0.463%.However,0.1%genipin did not significantly decrease this porosity(95.206 ± 0.663%,p > 0.05).As the concentration of genipin increased to 0.5% and 1%,the porosity of the scaffolds decreased to 92.503 ± 1.352% and 89.533 ± 0.794%,respectively.The scaffolds cross-linked with glutaraldehyde had the lowest porosity(86.791 ± 1.163%).At all of the time points,the release of hydroxyproline decreased as the concentration of genipin increased,indicating that cross-linking with genipin stabilized the 3D type ? collagen scaffold.Scaffolds cross-linked with glutaraldehyde showed the lowest release of hydroxyproline,while the non-cross-linked group demonstrated the highest hydroxyproline release.Compared with the non-cross-linked group,all of the cross-linked groups had significantly slower biodegradation(p < 0.01)at all time points.The group cross-linked with 0.1% genipin had the highest expression of Acan,Sox9,and Col2 compared with the other groups.The 0.5% genipin group demonstrated increased expression of Acan,Sox9,and Col2 compared with the non-cross-linked group.The 1% genipin group exhibited lower Col2 expression than the non-cross-linked group.3D type ? collagen scaffolds cross-linked with 0,0.1 and 0.5%genipin significantly increased Krt19(4.86,11.44,and 5.99-fold,respectively,compared with control group)and Pax1(12.43,18.00,and 6.43-fold,respectively,compared with control group)expression.The expression of all three gene markers significantly increased after ADSCs were cultured in the 3D type ? collagen scaffold cross-linked with 0.1% genipin.Conclusion:Cross-linking by genipin increased the stability of the type ? collagen scaffolds,but deformed the configuration of scaffolds and changed the intrinsic properties of type ? collagen.scaffold cross-linked with 0.1% genipin improved the biostability on the basis of maintaining the configuration of scaffold.In addition,the 0.1%genipin-cross-linked scaffold promoted ADSCs proliferation and differentiation into NP-like cells,along with the increasing gene and protein expressions of Sonic Hedgehog(Shh).All these results suggested that 0.1% genipin was the optimal concentration to establish a bio-stable 3D type ? collagen scaffold,which inducing ADSC proliferation and differentiation toward a NP-like phenotype through the activation of Shh signaling pathway.Part 2:Genipin-cross-linked cell delivery system for differentiation of adipose-derived stem cells and nucleus pulposus regenerationObjectives:Stem cell-based tissue engineering is a promising treatment for intervertebral disc(IVD)degeneration.A bio-scaffold that can maintain the function of transplanted cells and possesses favorable mechanical properties is needed in tissue engineering.Decellularized nucleus pulposus(d NP)has the potential to be a suitable bio-scaffold because it mimics the native nucleus pulposus(NP)composition.However,matrix loss during decellularization and difficulty in transplantation limit the clinical application of d NP scaffolds.Methods:In this study,we fabricated an injectable d NP-based cell delivery system(NPCS)and evaluated its properties by assessing the microstructure,biochemical composition,water content,biosafety,biostability,and mechanical properties.We also investigated the stimulatory effects of the bio-scaffold on the NP-like differentiation of adipose-derived stem cells(ADSCs)in vitro and the regenerative effects of the NPCS on degenerated NP in an in vivo animal model.Results:The results showed that approximately 68% and 43% of the collagen and s GAG,respectively,remained in the NPCS after 30 days.When the frequency was 0.1 or 1 rad/s,no significant difference was observed in the values of G' and G'' among fresh NP,d NP,and the 0.02% genipin-cross-linked NPCS.However,when the frequency was 10rad/s,the values of G' and G'' for d NP were significantly lower than those of the other groups.In addition,the 0.02% genipin-cross-linked NPCS and fresh NP had similar G'(13.71 ± 0.53 and 14.19 ± 0.59 k Pa,respectively)and G''(6.41 ± 0.15 and 6.56 ± 0.12 k Pa,respectively)values at 10 rad/s.0.02% genipincross-linked NPCS group had higher gene expression levels of Acan than the other two groups at day 7.There were no significant differences between the d NP and 0.02% genipin-cross-linked NPCS groups in the gene expression of Col2 and Sox9 at day 7.The gene expression levels of Acan,Col2,and Sox9 were significantly higher in the 0.02% genipin-cross-linked NPCS group than in the other groups at day 14.The disc height index(almost 81%)and the MRI index(349.05 ± 38.48)of the NPCS-treated NP were significantly higher than those of the degenerated NP after 16 weeks.The NPCS also partly restored the ECM content and the structure of degenerated NP in vivo.Conclusion:The NPCS showed mechanical properties similar to those of fresh NP.In addition,the NPCS was biocompatible and able to induce NP-like differentiation and extracellular matrix(ECM)synthesis in ADSCs.NPCS has good biological and mechanical properties and has the ability to promote the regeneration of degenerated NP.